Influence of a 50bp Ins/Del polymorphism at promoter of the superoxide dismutase-1 on gene expression and risk of heroin dependency
Saify and Saadat Environmental Health and Preventive Medicine
Influence of a 50bp Ins/Del polymorphism at promoter of the superoxide dismutase-1 on gene expression and risk of heroin dependency
Khyber Saify 0
Mostafa Saadat 0
0 Department of Biology, College of Sciences, Shiraz University , Shiraz 71467-13565 , Iran
Objective: Superoxide dismutase-1 (SOD1, OMIM: 147450) is one of the major antioxidant enzymes, which plays a vital role in clearance of reactive oxygen species. A genetic polymorphism of 50 bp insertion/deletion (Ins/Del) in the promoter region of the SOD1 was reported. The aims of the present study are to evaluate the influence of this polymorphism on the SOD1 mRNA levels in human peripheral blood cells and its association with risk of heroin dependency. Methods: The present study consisted of 47 healthy students of Shiraz University (south-west Iran) for investigating the association between the Ins/Del polymorphism on expression level of SOD1, also a total of 442 heroin dependent and 799 healthy controls were included in a case-control study investigating the association between the study polymorphism and risk of dependency to heroin. The quantitative SOD1 mRNA expression levels were investigated using quantitative real-time PCR. Results: Statistical analysis revealed a significant difference between the study genotypes (t = 5.17; df = 45; P < 0.001). The Del allele of the study polymorphism decreased approximately 33% of the SOD1 mRNA levels of the gene in the heterozygote individuals. Statistical analysis indicating that in both genders, neither the Ins/Del nor the Del/Del genotypes was associated with the risk of heroin addiction. Conclusions: The present study indicating that although the Ins/Del polymorphism of SOD1 is associated with the SOD1 expression levels, this polymorphism is not associated with the risk of dependency to heroin.
mRNA level; Ins/Del; Polymorphism; SOD1
Superoxide dismutase-1 (EC 126.96.36.199; SOD1, OMIM:
147450), a major cytoplasmic antioxidant enzyme is a
copper- and zinc-containing enzyme. The SOD1
metabolizes of highly reactive and more dangerous superoxide
radicals into less reactive molecular oxygen and
hydrogen peroxide (H2O2), thus providing a defense against
oxygen toxicity . It should be noted that the H2O2 is
subsequently converted into water by catalase or
glutathione peroxidase. Failure in the conversion processes or
imbalance between the production of reactive oxygen
species and the antioxidant enzymes, results in cell
damage, which leads to several multifactorial complex
traits such as cancers, psychiatry diseases, etc [2–5].
Several genetic polymorphisms in the SOD1 have been
reported in human populations (http://www.ncbi.nlm.nih.
studies have shown that SOD1 polymorphisms were associated
with the risks of senile cataract, cancers, etc [6–11]. A
genetic polymorphism has been reported in the promoter
region of SOD1 (1684 bp upstream of the ATG start codon)
. This is called 50 bp insertion/deletion (Ins/Del)
polymorphism. It has been shown that the 50 bp-deleted region
contains a number of transcription factor binding sites.
Studies indicate that the SP1 binds to this region. To
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determine if the 50 bp Ins/Del polymorphism affects the
basal transcription activity of the SOD1, an in vitro study
was published . Functional studies to assess the SOD1
promoter activity in several human cell lines transfected
with luciferase constructs, demonstrated that the Del allele
reduce the activity of the SOD1 promoter activity by
approximately 50% . In a more recent study, the 50 bp
deletion was found to be associated with a reduction in
SOD1 enzymatic activity in erythrocytes of control subjects
suggesting a moderate reducing effect on SOD1 synthesis
. The effect of this polymorphism on the mRNA levels
of the SOD1 was studied in the peripheral blood cells of 48
patients with sporadic amyotrophic lateral sclerosis (ALS)
and no difference was found among the genotypes
. There is no in vivo study on relationship
between the Ins/Del polymorphism and promoter
activity of the SOD1 in healthy subjects.
Opiates may cause oxidative stress in drug dependent
persons [15, 16]. It has been reported that morphine
decreases the mRNA level of mu-opioid receptor via
production of reactive oxygen species (ROS) in human
neuroblastoma SH-SY5Y cells . Oxidative stress is
very important in brain, because oxidative stress has
effects on the integrity of DNA as well as on the function
of the N-type and/or L-Type Ca2+ channel . On the
other hands, it has been demonstrated that SOD1 was
expressed in the various parts of brain, including motor
and sensory cranial nerve nuclei, as well as diffusely
through the brain in the neurons of the cortex, certain
regions of the hippocampus, and amygdale . Very
recently the alterations of the SOD1 mRNA levels in
SH-SY5Y human cells exposed to methadone and
morphine were reported [20, 21].
Although the association between GSTs
polymorphisms and susceptibility to drug dependency has been
reported [22–26], there are no study investigating the
association between the Ins/Del polymorphism of the
SOD1 and risk of heroin dependence. The aims of the
present study are to evaluate the influence of the Ins/Del
polymorphism on the SOD1 mRNA levels in human
normal peripheral blood cells and investigating the
association between this genetic polymorphism and risk of
The present study consisted of 47 (18 females, 29 males)
healthy students of Shiraz University (south-west Iran)
between the ages of 20 and 35 years (mean ± SD: 25.3 ±
3.3). A detailed description of these subjects has been
reported in our previous report . Considering that
Iranian population is a heterogeneous population [28–30],
we selected the participants from Persian (Caucasians)
Muslims living in Shiraz (Fars province, south-west Iran).
Informed consent was obtained from each volunteer
before the study. This study was approved by the Shiraz
University ethics committee.
Sample size calculation was undertaken using the
QUANTO (http://biostats.usc.edu/software) software. In
order to detect a real difference in mRNA levels between
the genotypes of the Ins/Del polymorphism with a
power of 0.90, α = 0.05, R2g (marginal proportion of
variance in the SOD1 mRNA levels explain by the 50 bp
Ins/Del polymorphism) = 0.25, 15% frequency of the
minor allele (the Del allele), a minimum sample of 27
would be necessary. Therefore, the present study is
more than sufficiently powered with an N = 47
subjects to detect a large effect in mRNA levels
between the different genotypes.
This report also consist a case-control study
performed in Shiraz (southern Iran) performed on 442
heroin dependent subjects (400 males, 42 females) and
799 healthy controls (662 males, 137 females). A detailed
description of these subjects has been reported in our
previous report . All patients were assessed using the
Structured Clinical Interview based on Diagnostic and
Statistical Manual of Mental Disorders, fourth edition
(DSM-IV) criteria for heroine and opium dependence.
Moreover, urine drug screens were obtained. All patients
were interviewed by a senior psychiatrist. The patients
were in methadone maintenance for treating heroin
dependency and all of them reported heroine as their
primary drug of choice. Control individuals were blood
donors, who declared that they did not suffer from
substance abuse. Using the QUANTO software, to detect a
real difference in genotypic frequency with a power of
0.80, α = 0.05, OR = 1.50, and 15% frequency of the Del
allele; a minimum sample of 331 would be necessary.
The present case-control study (with 1241 subjects) is
more than sufficiently powered to detect a
smallmedium effect in genotype frequency between cases and
Genotyping for the SOD1 Ins/Del polymorphism was
carried out using PCR based method, as described
previously . Total RNA was isolated from peripheral
blood cells by RNX-Plus kit (CinnaGene, Iran) following
the manufacturer’s instructions. RNA samples were used
for cDNA synthesis using the PrimeScript RT regent Kit
(Takara, Japan). Relative abundance of the SOD1 mRNA
level was assessed using quantitative real-time PCR
using a Rotor-gene 6000 real-time PCR system (Corbett
Life Science) by SYBR Green Premix Ex Taq II Kit
(Takara, Japan), as described previously . The “TATA
box-binding protein” (TBP, OMIM: 600075) was used as
calibrator gene. The SYBR green I fluorescence intensity
was acquired at the end of extension step of each cycle.
Relative differences in gene expression were expressed
using cycle threshold (Ct) values. ΔCt means difference
of Ct between TBP and SOD1. The SOD1 expression for
each individual was determining using 2-ΔCt .
Goodness-of-fit χ2 test was used to verify whether the
distribution of SOD1 genotypes was in accordance
with the Hardy-Weinberg equilibrium. The SOD1
expression levels were expressed as mean ± SE. To
evaluate the possible influence of the 50 bp Ins/Del
polymorphism in promoter region of the SOD1 on
the expression levels of the student t-test. In order to
exclude the possible influence of age and gender of
participants on the relationship between the SOD1
mRNA levels and the study polymorphism, multiple
regression analysis were used. In multiple regression
analysis the SOD1 polymorphism was included in as
number of the Del allele (0 and 1 used for Ins/Ins
and Ins/Del genotypes, respectively, gender was coded
as 0 for females and 1 for males and age of
participants (years) was used as a covariate.
The associations between the genotypes and the risk
of heroine dependence were assessed by odds ratios
(ORs) and 95% confidence intervals (CIs). Adjusted
ORs for age and gender of participants were also
estimated using logistic regression analysis. The
reference group consisted of individuals with the Ins/Ins
genotype. Statistical analysis was performed using the
Statistical Package for Social Sciences (SPSS Inc.,
Chicago, IL, USA; version 11.5). A probability of P < 0.05
was considered statistically significant.
Association between the Ins/Del polymorphism and SOD1
mRNA levels in healthy individuals
In our samples, 33 and 14 participants have the Ins/
Ins and Ins/Del genotypes, respectively. Genotypes in
the study group were in accordance with the
HardyWeinberg equilibrium (χ2 = 1.43, df = 1, P = 0.230).
Because of low prevalence of the Del allele, we did
not find the Del/Del genotype in our sample. The
frequency of the Ins/Ins genotype was similar between
female (0.66) and male (0.72) participants (χ2 = 0.17,
df = 1, P = 0.675). The age of participants with the Ins/Ins
(mean = 25.6, SD = 3.6 years) and Ins/Del genotypes
(mean = 24.6, SD = 2.1 years) was similar (t = 0.97, df = 45,
P = 0.333). Figure 1 show the SOD1 mRNA levels in
Ins/Ins and Ins/Del genotypes. Statistical analysis
revealed that there was a significant difference
between the Ins/Ins and Ins/Del genotypes for the
expression levels of SOD1 (t = 5.17, df = 45, P < 0.001).
Significant difference between the SOD1 genotypes
for the mRNA level was observed in multiple
regression analysis when age and gender of
participants were included in the analysis, as covariates
(Table 1). The present analysis indicated that the
50 bp deleted allele, down-regulates (approximately
33%) the expression levels of the SOD1 in the
Fig. 1 The SOD1 mRNA levels versus the TBP levels in the genotypes of the Ins/Del genetic polymorphism at promoter region of the SOD1 gene.
Data were shown as mean ± SE
Table 1 Association between the mRNA levels of SOD1 and the
50 bp Ins/Del genetic polymorphism at promoter region of the
SOD1 polymorphisma -3.340
Unstandardized Standardized t
Adjusted R2 for model = 0.350; F = 9.261; df = 3, 43; P < 0.001
aSOD1 polymorphism was included in analysis as number of the Del allele (0
and 1 used for Ins/Ins and Ins/Del genotypes, respectively)
bGender was coded as 0 for females and 1 for males
Association between the Ins/Del polymorphism and
dependence to heroine
Table 2 shows the genotypic prevalence of the study
polymorphism between the cases and healthy controls. The
genotypic frequencies of the polymorphism in healthy
controls (χ2 = 1.63, df = 1, P = 0.201) and patients (χ2 = 1.38,
df = 1, P = 0.239) were consistent with the Hardy-Weinberg
equilibrium distribution. Considering the significant
difference between genders for addiction risk and there are some
reports on the significant differences between genders for
the risks associated with drug dependency and genetic
polymorphism , the analysis were performed on each
gender group separately. Statistical analysis indicating that
in both genders, neither the Ins/Del nor the Del/Del
genotypes was associated with the risk of heroin addiction
(Table 2). The same results were observed after ORs were
adjusted for age and gender of participants (Table 2).
The present study indicated that the Del allele,
downregulates (approximately 33%) the expression levels of
the SOD1 in the heterozygote individuals, confirming
the previous studies which they determined the
expression levels of SOD1 in human cell lines transfected with
luciferase constructs  and the study reporting that
the 50 bp deletion was found to be associated with a
reduction in SOD1 enzymatic activity in erythrocytes of
control subjects . It should be noted that there is
another study that published in 2012 by Milani and
colleagues  that measured SOD1 mRNA levels in 48
ALS patients and they did not find a relevant difference.
The inconsistency between our present finding and the
finding of Milani and colleagues, at least in part might
be interpreted by this point that we used healthy
participants whereas they used ALS patients.
It has been shown that oxidative stress significantly
associated with several multifactorial diseases [2–5]. On
the other hands, based on the enzyme activity of SOD1
(converting highly toxic superoxide radicals into less
reactive molecules, hydrogen peroxide and oxygen)  it
is self-evident that this enzyme is potentially important
in etiology of several complex diseases. Considering that
the Del allele, leads to decreased of the activity of
promoter of the SOD1; subsequently it may alter the level
of ROS detoxification. Due to the high interaction of
ROS with DNA, the Ins/Del genetic polymorphism may
play an important role for inter-individual differences in
maintaining the genome’s integrity. Taken together, we
hypothesis that this polymorphism can modulate the risk
of dependency to heroine. However, the results of the
Table 2 Association between the 50 bp Ins/Del genetic polymorphism at promoter region of the SOD1 and risk of heroin
aAdjusted ORs for age and gender of participants
bAdjusted ORs for age of participants
present case-control study indicating that the risk of
heroin dependence is not associated with the Ins/Del
polymorphism of SOD1; therefore our hypothesis was
It has been reported by epidemiological studies that high
consumption of cruciferous vegetables would reduce
cancer risk [34–36]. On the other hand, alteration in
expression levels of SOD1 was reported when human hepatoma
cell line (HepG2-C8) was exposed to some common
phytochemicals present in cruciferous vegetables . It
should be noted that in the present study we did not ask
the participants about their environmental factors and life
style such as dietary consumption of cruciferous
vegetables. Simultaneous study the influence of the SOD1 Ins/
Del polymorphism and environmental factors on the
SOD1 mRNA level and risk of dependency to heroin
should be further researched. Our present study has
another limitation. We know that several polymorphisms
were reported in SOD1 (http://www.ncbi. nlm.nih.gov/
projects/SNP/snp_ref.cgi?geneId = 6647). The associations
between some of these polymorphisms and several
multifactorial traits were reported [6–11]. In the present study,
however, we study only the 50 bp Ins/Del in the promoter
region of SOD1.
The authors are indebted to the participants for their close cooperation. This
study was supported by Shiraz University (Grant number 89GCU1M1741).
All authors contributed significantly to this work. KS contributed to
experimental work and MS designed the experiments and analyzed the
results. Both authors reviewed the manuscript and approved the final draft.
The authors declare that they have no competing interests.
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